This application claims priority to European Pat. Appln. 98 105 043.8, filed Mar. 19, 1998.
The present invention relates to in vitro methods, kits and reagents for the qualitative screening and quantitative determination of the functional activity of components of the Protein C anticoagulant pathway of blood coagulation.
Maintenance of proper hemostasis is the result of a careful balance between procoagulant and anticoagulant activities. After a trauma, coagulation is triggered primarily through activation of coagulation Factors IX and X (FIX, FX) by tissue factor (also denoted tissue thromboplastin) and Factor VII (FVII) followed by generation of thrombin, which in turn cleaves fibrinogen to form soluble fibrin. After crosslinking by Factor XIII, a three-dimensional insoluble gel clot is obtained which prevents further blood losses.
Regulation of this highly potent system, shown schematically in FIG. 1, is accomplished by a balanced relation between procoagulant activities (shown with solid line arrows) and anticoagulant activities (shown with dashed line arrows). The anticoagulant activities include (1) inhibition of thrombin by antithrombin (AT) and xcex12-macroglobulin, and (2) prevention of further thrombin formation by the Protein C anticoagulant pathway. In that pathway, activated Protein C (APC) inactivates the coagulation proteins Factor VIII and Factor V in their activated forms (FVIIIa, FVa) through proteolytic cleavage. In addition, Factor Xa is inhibited by antithrombin and tissue factor pathway inhibitor (TFPI), the latter also inhibiting the tissue factor/Factor VIIa complex. Factor VIIa and Factor Va have potent procoagulant activities as cofactors in the activation of Factor X and prothrombin, respectively, and increase the reaction rates of these processes about 1000-fold each. Therefore, the inactivation of Factors Va and VIIIa by APC essentially stops further thrombin generation, thus providing a strong anticoagulant effect. Protein S and Factor V act as cofactors to activated Protein C (APC).
As shown in FIG. 1, the activation of coagulation through the intrinsic or extrinsic systems results in the activation of Factor X, a key component in the final common pathway. In the intrinsic system, the initial event is the activation of contact factors (Factor XII, prekallikrein) followed by the activation of Factor XI, which in turn activates Factor IX. In the extrinsic system, Factor IX and Factor X are activated by the tissue factor/Factor VIIa complex.
As FIG. 1 shows, calcium ions have to be present in several of these reactions. The activation of Factor X by Factor IXa, and of prothrombin by Factor Xa, also requires procoagulant phospholipids. In vivo, this is provided by the membrane surface of activated platelets; in vitro by platelet extracts, purified phospholipids, synthetic phospholipids and/or crude phospholipid extracts from suitable sources. The total and free calcium ion concentrations in native plasma are about 2.4 and 1.2 mmol/L, respectively. Typically, calcium ion concentrations used in analytical methods for the determination of coagulation or anticoagulation factors are in the range 1.5-10 mmol/L. The concentrations of other metal ions in plasma are lower, with typical values for the total concentration being 1 mmol/L for Mg2+ and 5-40 xcexcmol/L for Zn2+, Cu2+ and Mn2+.
Defects in the Protein C anticoagulant pathway may increase the risk of thrombosis due to a decreased capacity to prevent thrombin formation. Such defects may be due to deficiencies in the activity of Protein C and/or its cofactor Protein S. Another recently detected defect is a point mutation in the Factor V gene (Gxe2x86x92A) at nucleotide 1691, resulting in the amino acid substitution Arg (R)xe2x86x92Gln (Q) at position 506 in Factor V/Factor Va, denoted FV:Q506 or Factor V Leiden. Heterozygosity and homozygosity for this mutation are often denoted FV:R506Q and FV:Q506Q, respectively. This mutation is at one of the three APC cleavage sites (amino acids 306, 506, 679) in Factor Va, impairs its degradation by activated Protein C (APC), and confers a condition denoted as APC resistance.
APC resistance is to be considered a blood coagulation disorder recognized by an abnormally low anticoagulant response to activated Protein C (APC), and the determination of APC resistance may be used to screen for and diagnose thromboembolic diseases, such as hereditary thrombophilia, or for determining the risk for a human to acquire a manifestation of this blood coagulation disorder (e.g., European Pat. No. 608235).
Hence, there is a need to investigate these components of the Protein C anticoagulant pathway in the evaluation of thrombotic patients, and potentially also to screen for abnormalities of Protein C, Protein S and Factor V anticoagulant activity in situations connected with an increased risk of thrombosis, such as before surgery, during and after trauma, during pregnancy, or in connection with the use of oral contraceptive pills or hormone replacement therapy. Currently, clotting and/or chromogenic assays are available for analysis of Protein C and Protein S activity as well as for the detection of APC resistance (at least 90% of which is due to the FV:Q506 mutation).
Protein C activity is typically measured after activation of endogenous Protein C, contained in a plasma sample, by thrombin or by a snake venom enzyme from Agkistrodon contortrix contortrix (e.g., European Patent 203509 to Stocker), commercially available as the reagent Protac(copyright)C (Pentapharm AG, Basel, Switzerland). The concentration of Protac(copyright)C in the activation mixture is typically about 0.1 U/mL or higher since otherwise an insufficient activation of Protein C may be obtained (Martinoli et al. (1986), Thromb. Res. 43:253-264; McCall et al. (1987), Thromb. Res. 45:681-685).
After activation by Protac(copyright)C, the protein C activity is determined with a clotting or chromogenic assay (Bertina (1990), Res. Clin. Lab. 20:127-138; Marlar et al. (1989), Hum. Pathol. 20:1040-1047; European Pat. No. 486515). In clotting methods, coagulation is triggered through the intrinsic pathway by using APTT reagents or through the extrinsic pathway with the use of tissue factor. In both cases calcium ions are added to a final concentration of usually 5-10 mmol/L. Commercial kits and reagents are available for the determination of Protein C activity, such as Acticlot(trademark) C (American Diagnostica GmbH, Pfungstadt, Germany), Stachrom Protein C (Diagnostica Stago, Asnixc3xa8res, France), Staclot Protein C (Diagnostica Stago, Asnixc3xa8res, France), Coamatic(copyright) Protein C (Chromogenix AB, Mxc3x6lndal, Sweden) and Protein C Activator (Dade Behring, Deerfield, Ill.).
The activation of Protein C by thrombin is stimulated about 1000-fold by thrombomodulin, an endothelial cell membrane protein (Esmon et al. (1981), Proc. Natl. Acad. Sci. (USA) 78:2249-2252). The use of thrombin/thrombomodulin as activator of Protein C for analysis of Protein C and/or Protein S activity in plasma samples, utilizing a photometric method, is also known (French Pat. Appln. No. 2689 640-A1).
Protein S activity is determined from its stimulation of the activity of APC in its degradation of Factor Va and/or Factor VIIIa. Typically, in such assays a standardized amount of APC is added to a plasma sample or activation of endogenous protein C is performed whereafter the clotting time is determined after a simultaneous or separate coagulation activation via the intrinsic system using an APTT reagent, via the extrinsic system using tissue factor or Factor Xa (Bertina (1990), supra; Preda et al. (1990), Thromb. Res. 60:19-32; D""Angelo et al. (1995), Thromb. Res. 77:375-378). Chromogenic activity assays for protein S have also been published, utilizing Factor IXa as an activator and monitoring Factor Xa generation (European Pat. No. 567 636) or thrombin generation (European Pat. No. 486 515). In all these methods, calcium ions are added as mentioned above.
The FV:Q506 mutation in the Factor V molecule may be detected with molecular biology methods based upon the use of the polymerase chain reaction (PCR) technique (Bertina et al. (1994), Nature 369:64-67), or by methods in which the functional activity of APC is determined. Such functional activity methods may be coagulation-based (e.g., European Pat. No. 608235; Rosxc3xa9n et al. (1994), Thromb. Haemost. 72:255-260), and may include the use of predilution of sample plasma with a plasma with little or no Factor V activity (European Pat. Appln. No. EP-A-94 905 908.3; Jorquera et al. (1994), Lancet 344:1162-1163; Svensson et al. (1997), Thromb. Haemost. 77:332-335). The latter assay principle, a coagulation-based assay using predilution of sample plasma, is also utilized in a commercial product, Coatest(copyright) APC Resistance V (Chromogenix AB). Alternatively, chromogenic methods may be used (European Pat. No. 608 235; Rosxc3xa9n et al. (1995), Thromb. Haemost. 73:1364, Abstract 1778; Nicolaes et al. (1996), Thromb. Haemost. 76:404-410).
Since genetic defects in the Protein C anticoagulant pathway are found in about 25% of unselected patients with venous thromboembolism (VTE) and in about 50% of patients with thrombophilia (i.e., patients from families with an increased tendency to VTE), there is a need for a single test which detects all such abnormalities with a high sensitivity and specificity, i.e., a global (overall) test. One concept for a global test is based upon the activation of Protein C in plasma with Protac(copyright)C and activation of coagulation via the intrinsic or extrinsic pathway (U.S. Pat. No. 5,001,069; European Pat. Appln. No. 696 642). Results obtained with a commercial kit application of this test, ProC Global (Behring Diagnostica, Marburg, Germany), in which intrinsic activation of coagulation is accomplished through addition of an APTT reagent, show a sensitivity for Protein C deficiency, Protein S deficiency, and the FV:Q506 mutation of, respectively, about 90%, 50-80% and more than 90% on analysis of healthy individuals and thrombotic patients (Dati et al. (1997), Clin. Chem. 43:1719-1723; Ruzicka et al. (1997), Thromb. Res. 87:501-510). The specificity, however, is about 50% in thrombotic cohorts and, therefore, a substantial proportion of positive results are obtained which can not be linked to known defects in components of the Protein C anticoagulant pathway, such as in protein C, protein S and Factor V. Thus, this test lacks sufficient specificity.
Furthermore, results from analysis of pregnant women lacking any of the known defects in the Protein C anticoagulant pathway are clearly different from analysis of normal healthy individuals (Rangxc3xa5rd et al. (1997), Annals Hematol. 74, Supplement II, Abstract 74, A77; Siegemund et al. (1997), Annals Hematol. 74, Supplement II, Abstract 188, A105), which necessitates separate ranges for these cohorts. This as yet uncharacterized interference limits the general applicability of the test. An alternative global method for the detection of defects in the protein C anticoagulant pathway, based upon activation of endogenous plasma Protein C by Protac(copyright)C utilizes tissue factor as the trigger ofthe coagulation (Preda et al. (1996), Blood Coag. Fibrinol. 7:465-469). The sensitivity of this method also is limited, especially for Protein S. Furthermore, different sample categories, e.g., pregnant and non-pregnant women, may require different approaches for evaluation of the results due to interference from factors not related to known defects of the Protein C anticoagulant pathway.
For a global test to be useful as a screening test for known inherited defects in the Protein C anticoagulant pathway (e.g., Protein C deficiency, protein S deficiency or the FV:Q506 mutation), the sensitivity should be high, at least 90%, for all these defects. Furthermore the specificity should be high, above 60%, preferably above 70%, and more preferably above 80%, in order to considerably reduce the number of false positive results. The state-of-the-art methods do not provide a satisfactory solution to these requirements. For the development of improved methods for the specific determination of Protein C and Protein S activity, and for determination of mutations in Factor V which affect its anticoagulant activity, it is also desirable to improve the resolution and specificity of these methods. There is also a need to improve the stability of different reagents used in such methods.
Thus, the technical problem underlying the present invention is the provision of in vitro methods with improved sensitivity and specificity for diagnostic screening and for the specific detection of defects in the Protein C anticoagulant pathway in humans. A further recognized problem is to improve the stability of reagents used in such methods.
The present invention is based, in part, upon the surprising finding that the addition of low levels of divalent metal ions, such as Mg2+, Mn2+, Zn2+, Ni2+, Sr2+, or Cu2+ ions, or of the monovalent copper ion (Cu+), in the presence of calcium ions, enhances the anticoagulant activity of the Protein C anticoagulant pathway, provides for a high degree of resolution between different levels of Protein C activity and Protein S activity, provides a high degree of discrimination for the presence of the FV:Q506 mutation, and results in an improved sensitivity and specificity for detection of defects in components of the Protein C anticoagulant pathway with photometric and/or clotting methods. Thus, the invention also constitutes a xe2x80x9cglobalxe2x80x9d method for detecting defects in the Protein C anticoagulant pathway. In addition, the addition of divalent metal ions provides for an unexpected improvement of the stability of the reagents typically used in determining the anticoagulant activity of components of the Protein C anticoagulant pathway.
Thus, in one aspect, the present invention provides an in vitro method for qualitative screening and quantitative determination of the functional activity of components of the Protein C anticoagulant pathway of blood coagulation, comprising measuring the conversion rate of an exogenous substrate by an enzyme, the activity of which is related to Protein C anticoagulant activity, in a human blood or plasma sample comprising coagulation factors and said exogenous substrate, after at least partial activation of coagulation through the intrinsic, extrinsic or common pathways and triggering coagulation by adding calcium ions; and comparing said conversion rate with the conversion rate of a normal human blood or plasma sample determined in the same way, said method being characterized by adding further metal(s) ions to said sample.
The present invention is thus concerned with in vitro methods for screening for, in a human, defects in the Protein C anticoagulant pathway due to, for example, Protein C deficiency, Protein S deficiency, or Factor V mutations such as the FV:Q506 mutation, or other Factor V defects related to APC resistance and/or APC cofactor activity. Such methods may be designed for the specific detection of Protein C deficiency, Protein S deficiency, or mutations in Factor V/Factor Va which affect the cleavage rate by APC. One preferred embodiment of the present invention comprises a global test for the Protein C anticoagulant pathway.
The methods of the invention allow improved screening and diagnosis of defects in the Protein C anticoagulant pathway in patients with thromboembolic diseases such as deep venous thrombosis and/or pulmonary embolism. In cases where a patient belongs to a family with hereditary thrombophilia, the methods are also suitable for the investigation of family members of the patient to determine the possible inheritance of defects within the pathway. The methods are also particularly useful for diagnosing defects in the Protein C anticoagulant pathway in patients before surgery, patients with trauma, in pregnant women, or in women receiving oral contraceptive pills or hormone replacement therapy such as estrogen therapy. Furthermore, the xe2x80x9cglobalxe2x80x9d methods of the invention may be used for the detection not only of known defects in the Protein C anticoagulant pathway, but also of hitherto unrecognized defects. In particular, the invention provides for specific photometric and/or clotting methods for such unrecognized defects in the Protein C anticoagulant pathway.
The methods of the invention may comprise monitoring the conversion of an exogenous photometric substrate for either Factor Xa or thrombin, containing a chromophore, fluorophore or luminophore as a leaving group. Examples of such photometrically measurable leaving groups are p-nitroaniline (a chromophore) for use in colorimetric methods; naphthylamine and coumarin derivatives such as methylcoumarine, aminoisophthalic acid and its derivatives (fluorophores) for use in fluorimetric methods; and isoluminolamide (a luminophore) for use in luminometric methods.